In the third phase of ChIP
we will use a Novus Antibody
that is specific for our protein of interest
to pull down DNA that is cross linked to that protein.
This is done by using magnetic beads
conjugated to a protein A/G
that will bind to the antibody/protein/
DNA complex.
Start by diluting samples 1 to 10
by adding 40 microliters of sheered chromatin
to 360 microliters of the IP dilution buffer and include
protease inhibitors.
All buffers are detailed in written protocols found in the support section
of the Novus website.
It is beneficial to IP at least three samples.
One will be for your antibody of interest,
another will be for a positive control antibody
and a third for a negative control which will not contain any antibody and will be
used to measure background levels.
If you are using the ChromataChIP Kit, your kit includes a positive control
antibody.
Dilute one last sample and label this input.
This sample will not go through the IP process and instead be saved for Phase Four
of the protocol.
Store this at four degrees Celsius until ready for use.
Add your antibody of interest and positive control antibody to the
respective diluted samples
then incubate overnight at four degrees Celsius with rotation.
The next day,
for each sample to be IPed
you will use 25 microliters of micro fully re-suspended protein A/G
magnetic beads.
First wash the beads by adding one mL of IP dilution buffer
to the amount of beads that you will be using for all of your samples
and then vortex.
Then pellet the beads with a magnetic separator
and re-suspend the beads with the same volume of IP dilution buffer
as your original volume of beads.
Add 25 microliters of wash beads to each sample
and incubate tubes with rotation for one hour at 4 degrees Celsius.
Next we can begin our wash steps.
Wash steps can be performed manually through the use of the rotator and
magnetic separation rack
or automated with a machine designed for this use.
Start by pelleting the beads and removing the supernatant.
Then add 500 microliters of cold IP wash buffer one
and wash for 5 minutes at 4 degrees Celsius with rotation or
agitation.
Repeat this entire step for IP wash buffers 2, 3, and 4.
Elute the complex from the beads by adding 200 microliters of
IP elution buffer and washing for 15 minutes at 4 degrees
Celsius with rotation or agitation.
Pellet the beads
keeping the supernatant
and discarding the beads.